Three homoisoflavonoids, including a new compound, 5,7-dihydroxy-3-(4-methoxybenzyl)-8-methyl chroman-4-one (1), together with two known compounds, 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-8-methylchroman-4-one (2) and 5,7-dihydroxy-3-(2-hydroxy-4-methoxybenzyl)-chroman-4-one (3), were isolated from the rhizomes of Polygonatum verticillatum (L.) All. (P. verticillatum). Isolated compounds were characterized on the basis of UV, FT-IR, ESI-MS, and 1D-, 2D-NMR data. Further, different extract fractions and pure compounds from Polygonatum verticillatum were screened for their antimicrobial potential. Among three pure compounds, compound 2 was found most potent with good zone of microbial growth inhibition as compared to the standards.